The mammalian cdc2 protein kinase: mechanisms of regulation during the cell cycle

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1 J. Cell Sci. Suppl. 12, (1989) Printed in Great Britain The Company of Biologists Limited The mammalian cdc2 protein kinase: mechanisms of regulation during the cell cycle GIU LIO DRAETTA a n d DAVID BEACH Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, New York 11724, USA Summary Recent experimental evidence has demonstrated the central role of the cdc2 protein kinase in the transition from G 2 to M phase in eukaryotic cells. We shall review our knowledge of the mechanisms which coordinate activation of the kinase with cell cycle-specific events in mammalian cells. The origins: cdc2+ and the fission yeast cell cycle In the fission yeast Schizosaccharomyces pombe the cell cycle is regulated at two steps, one acting in G i before D N A synthesis, the other in G 2 (Fantes and Nurse, 1977; Nasmyth, 1979). In the mid 1970s a number of cell cycle control genes were identified. Among them cdc2+ was particularly interesting because its function is required in G j, as well as in G 2 for the initiation of mitosis (Nurse and Bissett, 1981). In addition to lethal temperature-sensitive mutations in the cdc2+ gene, dominant wee mutations were described, which cause cells to undergo mitosis prematurely (Nurse, 1975). Therefore, cdc2+ acts at a rate-limiting step, which integrates cellular informations (cell size, completion of D N A synthesis etc.) and determines the timing of mitosis. cdc2+ is homologous to a previously described Saccharomyces cerevisiae cell cycle gene, CDC28. The two genes are functionally interchangeable: CDC28 can rescue S. pombe cdc2 mutations and a cdc2+ intronless gene can rescue cdc28 mutations of S. cerevisiae (Beach et al. 1982; Booher and Beach, 1986). The two genes both encode 34K (K = 103ilfr) polypeptides, which are 62% identical at the amino acid level and contain regions that are highly conserved among the family of Ser/Thr kinases. In addition both proteins have kinase activity in vitro (Reed et al. 1985; Simanis and Nurse, 1986; Brizuela et al. 1987). Given the universality of the basic organization of the cell cycle in all eukaryotes (ordered phases of D N A synthesis and nuclear division) it was legitimate to guess that cell cycle control elements might be conserved throughout evolution. A homologous cell cycle control kinase is, after all, present in two evolutionary very distance species (S. pombe and 5. cerevisiae). Based on this reasoning, an investigation into cell cycle control genes in higher eukaryotes was initiated. Key words: cdc2, tyrosine phosphorylation, protein kinase.

2 G. Draetta and D. Beach 22 The com ponents of the cdc2 com plex and their regulation (1) p34cdc2 Using monoclonal antibodies against fission yeast cdc2 which cross-reacted with the S. cerevisiae CDC28 protein, we identified a 34K protein kinase in human HeLa cells (Draetta et al. 1987). Proteolytic mapping, kinase activity of the immunoprecipitates and association of the human protein with the homolog of the s u c l + gene product of yeast (see below), unequivocally confirmed that a human cdc2 homolog had been found. A human cd N A sequence that was able to complement a temperature-sensitive mutation of cdc2+ in fission yeast was also cloned and sequenced (Lee and Nurse, 1987). This sequence showed 62% similarity to the fission yeast cdc2 sequence. Insight into the possible role of human cdc2 in cell cycle regulation was gained through investigation of the properties of cdc2 at different stages of the cell cycle (Draetta and Beach, 1988). As in fission yeast, the levels of the protein in the cell are constant during the cell cycle. However, phosphorylation of cdc2 appears to be cell cycle-regulated (Fig. 1). In G j cdc2 is dephosphorylated and phosphate accumulates on the protein as cells progress through S and G 2. Monomeric unphosphorylated cdc2 is inactive, as a protein kinase, whereas phosphorylated cdc2 is able to phosphorylate the artificial substrate casein in vitro (Draetta and Beach, 1988; Brizuela et al. 1989). Anaphase - Anaphase M etaphase -> Interphose M etaphase In te rp h a se Fig. 1. Model of the activation of the cdc2 kinase complex. In G i, cdc2 is unphosphoryl ated. During S and G 2, progressive accumulation of cyclin and phosphorylation of the cdc2 subunit leads to complex formation. At mitosis, cdc2 becomes dephosphorylated and it is maximally active. According to this model, phosphorylation of the cyclin subunit is the signal for its degradation and exit from mitosis.

3 Cell cycle of mammalian cells 23 The most highly phosphorylated form of cdc2 is associated with a 62K protein (p62, probably corresponding to human cyclin B; see below, and Fig. 1). Dephosphorylation of cdc2 protein when it is complexed with p62 probably triggers maximal activation of the kinase against endogenous p62 and added histone-hj or casein. Interestingly, cdc2 is phosphorylated in vivo not only on threonine and serine but also on tyrosine residues. Using anti-phosphotyrosine antibodies we found that, phosphotyrosine progressively accumulates on cdc2 as cells move from G i to S and G 2 (Draetta et al. 1988), and is lost at mitosis. Recently cdc2 was found to be a component of the M phase-specific histone-hj kinase of starfish oocytes (Arion et al. 1988; Labbé et al. 1988). This enzyme is a cyclic AM P and calmodulin-independent protein kinase, with a high affinity for histone-hi. Its cyclic activation at mitosis had suggested a possible involvement in cell cycle regulation. Also, the possibility of mediating chromosome condensation through phosphorylation of histone-hj had been suggested. Purified preparations of starfish histone-hi kinase contain equimolar amounts of cdc2 and a B-type cyclin, as described by Dorée et al (this volume). A cdc2 homolog is a component of the maturation-promoting factor (MPF) of Xenopus eggs (Dunphy et al. 1988; Gautier et al. 1988). MPF is a cellular factor that is able to induce meiotic maturation when injected in Xenopus oocytes that are arrested at the G 2/prophase boundary of the first meiotic division (Masui and Markert, 1971; Smith and Ecker, 1971). MPF activity has been detected in oocytes from a number of different vertebrate and invertebrate species. In addition, MPF can be detected in mitotic cells from sea urchin and amphibian embryos, as well as in yeast and mammalian cells (Kishimoto, 1988). (2) p!3 s cl su c l+ was isolated as a D N A sequence that rescues some but not all temperaturesensitive alleles of cdc2 when expressed on a multicopy plasmid. Disruption of the su c l+ gene causes cell cycle arrest, confirming a direct involvement of this gene in cell cycle control (Hayles et al. 1986; Hindley et al. 1987). The D N A sequence of the su c l+ gene encodes a protein of 113 amino acids (Hindley et al. 1987), which has been identified in yeast. Its abundance does not alter during the cell cycle or during entry into stationary phase (Brizuela et al. 1987). p 13 is complexed with the cdc2 gene product and the pl3sucl-p34cdc2 complexes have kinase activity. In addition, the stability of cdc2/sucl complexes is altered in strains carrying temperaturesensitive alleles of cdc2 which are suppressible by overexpression of the su c l+ gene (Brizuela et al. 1987). Kinase activity can be restored in vitro by addition of purified p 13 to the assay (Booher et al. 1989). In human cells, we have detected a homolog of the su c l+ protein. (Draetta et al. 1987). As in fission yeast, this protein is complexed with the cdc2 protein. In addition, recombinant yeast pl3sucl expressed in Escherichia coli is able to bind the cdc2 kinase in vitro and such complexes possess very high kinase activity. pl3sucl is able to bind both the p34cdc2 monomer and the cdc2 high molecular weight complex (Brizuela et al. 1989). P13 is neither a substrate nor an inhibitor of the cdc2 kinase.

4 24 G. D raetta and D. Beach At present we do not have any direct evidence for the role of the pl3 protein in the regulation of the cdc2 kinase complex. (3) p62 (cyclin B) p62 was identified as a component of the human cdc2 complex (Draetta and Beach, 1988). Immunoprecipitates from 35S-labeled or 32P-labeled HeLa cells with anticdc2 antibodies show a 62K band in addition to the p34 and p 13 bands. We have demonstrated a specific interaction between p34 and 62K species (Draetta and Beach, 1988; Brizuela et al. 1989). As already mentioned, p34 must be phosphorylated in order to interact with p62. p62 is a substrate of the cdc2 kinase in vitro. Using the phosphorylation of p62 in immunoprecipitates we have recently purified the human mitotic cdc2 complex (Brizuela et al. 1989). The sudden loss of activity of the cdc2 kinase at the end of mitosis and the concomitant loss of p62 from the complex suggested the possibility that the 62K protein might be a mitotic cyclin. The cyclins are proteins originally identified in clam and sea urchin eggs, which are synthesized after fertilization, accumulate across each cell cycle and are abruptly degraded at the end of each mitosis (Rosenthal et al. 1980, 1983; Evans et al. 1983). Injection of cyclin A or B m RN A into unfertilized Xenopus oocytes leads to resumption of the meiotic divisions and appearance of M PF activity (Swenson et al. 1986; Pines and Hunt, 1987; Westendorf et al. 1989). In a recent paper (Draetta et al. 1989) we have demonstrated a direct biochemical interaction between cdc2 and the clam cyclin A and B. In the clam Spisula solidissima we have identified a 34K cdc2 homolog. Together with cdc2, cyclin A and B are bound to pl3-sepharose. The pl3-bound complex as well as anti-cyclin A or B immunoprecipitates show histone-hi kinase activity. The kinase activity is only present in precipitates prepared from M phase but not interphase egg extracts. A human cyclin B clone has recently been identified (Pines and Hunter, 1989). Antibodies against a bacterially expressed cyclin B recognize the HeLa p62 (Draetta et al. unpublished). p62 is therefore the human homolog of cyclin B. The S. pombe cd cl3 + gene encodes a cyclin B homolog (Solomon et al. 1988; Goebl and Byers, 1988). Its gene product forms a stable complex with the fission yeast cdc2, and is an endogenous substrate of the kinase complex (Booher and Beach, 1988; Booher et al. 1989). As in human cells this complex shows cell cycle-regulated histone-hi kinase activity. InXenopus extracts, ablation of cyclin m RN A also blocks M PF activity (Minshull et al. 1989). In addition, it has been recently demonstrated that cyclin B m RN A is the only message needed to be translated in order to induce interphase Xenopus extracts to undergo M phase (Murray and Kirschner, 1989; Murray et al. 1989). cdc2 regulation: solved and unsolved matters During the first two years following the identification of a mammalian cdc2 homolog we developed a number of reagents that have advanced our knowledge of the regulation of the cdc2 kinase in mammalian cells. The observations in human cells

5 Cell cycle of mammalian cells 25 have strongly reinforced the idea of a direct involvement of cdc2 in mitotic control. Polyclonal and monoclonal antibodies produced against the yeast cdc2 protein, an anti-peptide serum which is able to recognize multiple phosphorylated forms of the cdc2 protein, and the recombinant pl3 protein have been utilized to produce our second generation cdc2 reagents. Purified preparations of the cdc2 monomer as well as of the complex are now available (Draetta et al. 1988; Brizuela et al. 1987) and they will allow us to address the following new and interesting questions. (1) What is the structure of the cdc2 complex at mitosis (and, just as important, what is the structure of the mammalian MPF)? Are there different mitotic-specific cdc2 kinases? (2) What are the kinases that phosphorylate cdc2? Is cdc2 a substrate of the weel kinase (the existence of w e e l+ homologs in species other than fission yeast is strongly suggested by the experiments of Russell et al. 1989). What are the tyrosine kinases that phosphorylate cdc2? Is their activity cell cycle-regulated? What is the nature of the phosphatases involved? What is the sequence of the phosphorylation events? (3) What is the role of the fission yeast cdc25+ homolog in the activation of mitosis (Russell et al. 1989); does it show a specific interaction with the cdc2 complex? (4) Is cdc2 phosphorylation or cyclin formation the rate-limiting step in the complex assembly in vivo? (5) What is the role of the pl3 protein? It is not a substrate nor an inhibitor/activator in vitro. Could it be a docking factor, which drives the cdc2 complex to some cellular components? (6) What are the cdc2 substrates? The study of the phosphorylation of the endogenous p62 cyclin B subunit will provide us with a starting point for the characterization of other cdc2 substrates, at least in terms of their biochemical requirements. Histone-Hi, which has a very low K m for cdc2, might be a physiological substrate. In fact, sites phosphorylated in vivo at mitosis are also phosphorylated in vitro. It is clear that given the large number of proteins that are phosphorylated at mitosis two types of hypothesis can be formulated, one in which cdc2 acts as the master kinase, catalyzing the phosphorylation of all substrates directly. The other cascade model would predict a number of more specific protein kinases, which are themselves substrates for cdc2 and catalyze - phosphorylation of specific subcellular components. In conclusion, we would like in the not too distant future to be able to use purified cellular components and reproduce in vitro the events that lead to mitosis in eukaryotes. Together with yeast genetics and molecular biology, and the study of the biology of MPF in early embryos, the newly born mammalian cdc2 biochemistry will probably add new elements to this goal. References A r io n, D., M eijer, L., Brizu ela, L. and Beach, D. (1988). cdc2 is a component of the M phase specific histone-histone-h! kinase: evidence for identity with M PF. Cell 55, Beach, D. H., D urkacz, B. and N urse, P. M. (1982). Functionally homologous cell cycle control genes in budding and fission yeast. Nature, Lond. 300, Booher, R. and Beach, D. (1986). Site-specific mutagenesis of cdc2+, a cell cycle control gene of the fission yeast Schizosaccharomyces pombe. Molec. cell. Biol. 6, Booher, R. and Beach, D. (1988). Involvement of cdcl3+ in mitotic control in Schizosaccharo-

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